DESCRIPTION (Adapted from applicants abstract): The direct binding of streptococci to human platelets is a postulated central mechanism in the pathogenesis of endocarditis. Bacterium-platelet binding may be critical for the attachment of blood-borne organisms to the valve surface, and for the subsequent formation of infected, macroscopic vegetations. The aim of this project is to define the molecular basis for the direct binding of Streptococcus sanguis to human platelets, and to determine the role of binding in the pathogenesis of endocarditis. By means of transposon mutagenesis, four isogenic mutants of Streptococcus sanguis strain M99 have been generated that bind platelets minimally in vitro. These mutants will provide a basis for the proposed research. A putative streptococcal gene ("spl~) encoding a ligand for human platelets will be identified, using probes derived from the low-binding mutants to screen a genomic library of strain M99, followed by cloning and sequencing. The spl gene product will then be purified, by cloning into a pET vector expression system. Once isolated, binding of the putative ligand to washed human platelets in vitro will be examined, using Scatchard analysis to determine if binding resembles a receptor-ligand interaction. The role of ligand mediated binding in the pathogenesis of endocarditis will be addressed in an animal model, by comparing the relative virulence of parental M99 with the isogenic mutant containing a Tn916deltaE insertion within the spl locus. By defining the mechanisms for streptococcal-platelet binding at the molecular level, this work will provide a basis for determining the role of platelets in the pathogenesis of endocarditis. In turn, this may provide a basis for developing novel diagnostic and therapeutic strategies.